Novel ethanol producing bacterium and process for producing ethanol

ABSTRACT

A bacterium capable of producing ethanol at a temperature of 40° C. or more from a carbon compound which is gaseous at ordinary temperature and ordinary pressure as the raw material, and a process for producing ethanol by culturing the bacterium at 40° C. or more.

TECHNICAL FIELD

The present invention relates to a bacterium capable of producingethanol at a high temperature from a gaseous carbon compound such ascarbon dioxide as the raw material, and a process for efficientlyproducing ethanol from the same.

BACKGROUND OF THE INVENTION

Since there is a background that the exhaustion of petroleum, the carbondioxide problem accompanied by greenhouse effect and the like, variousenergy sources have been developed. Particularly, as the movable bodyenergy to be used in transportation and the like, development of a novelliquid fuel considered to be relatively easily introduced in view of theuse of existing facilities and safety is drawing attention. Examples ofthe liquid fuel production processes so far developed include productionof a hydrocarbon from natural gas, production of methanol from syntheticgas, liquefaction of coal, production of ethanol from molasses andstarch. Among these, since octane number is high, ethanol is used as agasoline base material in foreign countries, and its application isexpected also in Japan.

The ethanol production so far employed is its fermentation productionwhich uses microorganisms, and field crops such as sugar cane and cornare used as the raw material. Although it is necessary to produceethanol at a moderate price when used as a fuel, inexpensive ethanolproduction is considerably difficult to carry out by the conventionalproduction process, since the raw material competes with food and theraw material becomes expensive. Thus, development of ethanol productiontechniques which use a cheap raw material has been desired in recentyears. One of them is a process in which cheap organic waste such as anagricultural waste and waste woodare used as the raw material andsubjected to a saccharification treatment by an acid or enzyme, andethanol is produced by fermenting the solution. Although production ofethanol at a moderate price can be expected by this process, since theraw material is a waste, there are many problems to be solved in view ofcost and techniques, such as the requirement of secondary treatmentfacilities for the waste water treatment of treated water generated atthe saccharification step and high load water after the culturing, sothat the development of an ethanol production technique which using nosugar as the raw material has been desired.

As a countermeasure, development of an ethanol production techniqueusing gas such as carbon monoxide and carbon dioxide as the raw materialhas been proceeded. In this technique, ethanol is produced from a gasobtained by burning or the like as the main material, using amicroorganism which can use the gas. Clostridium ljyungdahlii (U.S. Pat.No. 5,173,429 (1992)) and Clostridium autoethanogenum sp. (Jamal Abriniet al., Arch. Microbiol., 161: 345-351 (1994)) are so far known asmicroorganisms which produce ethanol from a gas as the raw material. Itis reported in each case that growth and ethanol production are carriedout at 37° C. from a carbon monoxide-containing gas as the raw material.

However, when ethanol is produced by using the microorganisms, gas suchas carbon monoxide and carbon dioxide to be used as the raw material isproduced at high temperature and high pressure conditions in most cases.Accordingly, when a bacterium which grows and produces ethanol at 37° C.is used, gas-cooling facilities such as a heat exchanger and a largetank are required, so that a huge cost is required in the facility pointof view. Also, since the produced ethanol is toxic for the bacterium,its continuous production by culturing while taking out it from theculture is considered to be an efficient production process, but thebacteria used so far have a problem that large energy is consumed fortaking out the product because of the low culturing temperature andethanol production temperature, so that the low production temperatureis disadvantageous for the industrial production application.Accordingly, a bacterium which produces ethanol at higher temperaturefrom a gas as the raw material has been expected.

In addition, while a carbon monoxide-containing gas is used as the rawmaterial in the conventional techniques as described in the above, agreen house gas reducing effect can be expected in case that it ispossible to obtain a bacterium which can produce ethanol from carbondioxide as the raw material which is one of the green house gases.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a bacterium which canproduce ethanol in high efficiency at a higher temperature from gas suchas carbon dioxide as the raw material.

Another object of the present invention is to provide a process forefficiently producing ethanol using the bacterium at a high temperaturefrom a gas as the raw material.

The present invention relates to the following (1) to (17).

(1) A bacterium capable of producing ethanol at a temperature of 40° C.or more from a carbon compound which is gaseous at ordinary temperatureand ordinary pressure as the raw material.

(2) The bacterium according to (1), wherein the carbon compound is atleast one selected from the group consisting of carbon monoxide, carbondioxide, methane, ethane and ethylene.

(3) The bacterium according to (1) or (2), wherein the carbon compoundis at least one selected from the group consisting of carbon monoxideand carbon dioxide.

(4) The bacterium according to any one of (1) to (3), which belongs tothe genus Clostridium or a derivative genus thereof.

(5) The bacterium according to (4), wherein the genus Clostridium or aderivative genus thereof is the genus Clostridium, the genusThermoanaerobacterium, the genus Thermoanaerobacter or the genusMoorella.

(6) The bacterium according to any one of (1) to (5), which belongs tothe genus Clostridium.

(7) The bacterium according to any one of (1) to (6), which isClostridium sp. No. 16-1 (FERM BP-8372), Clostridium sp. No. 16-2 (FERMBP-8373), Clostridium sp. No. 22-1 (FERM BP-8374) or an analogousbacterium thereof.

(8) A process for producing ethanol, which comprises culturing, at atemperature of 40° C. or more, a bacterium capable of producing ethanolat a temperature of 40° C. or more from a carbon compound which isgaseous at ordinary temperature and ordinary pressure as the rawmaterial.

(9) The process according to (8), wherein said culturing is carried outin the presence of a carbon compound which is gaseous at ordinarytemperature and ordinary pressure.

(10) The process according to (8) or (9), wherein the carbon compound isat least one selected from the group consisting of carbon monoxide,carbon dioxide, methane, ethane and ethylene.

(11) The process according to any one of (8) to (10), wherein the carboncompound is at least one select-ed-from the group consisting of carbonmonoxide and carbon dioxide.

(12) The process according to any one of (8) to (11), wherein thebacterium belongs to the genus Clostridium or a derivative genusthereof.

(13) The process according to (12), wherein the genus Clostridium or aderivative genus thereof is the genus Clostridium, the genusThermoanaerobacterium, the genus Thermoanaerobacter or the genusMoorella.

(14) The process according to any one of (8) to (13), wherein thebacterium belongs to the genus Clostridium.

(15) The process according to any one of (8) to (14), wherein thebacterium is Clostridium sp. No. 16-1 (FERM BP-8372), Clostridium sp.No. 16-2 (FERM BP-8373), Clostridium sp. No. 22-1 (FERM BP-8374) or ananalogous bacterium thereof.

(16) The process according to any one of (8) to (15), which furthercomprises:

vaporizing the produced ethanol to thereby separate it from the culture;and

liquefying the separated ethanol.

(17) A process for producing ethanol, which comprises:

culturing, at a temperature of 40° C. or more, a bacterium capable ofproducing ethanol at a temperature of 40° C. or more from a carboncompound which is gaseous at ordinary temperature and ordinary pressureas the raw material;

vaporizing the produced ethanol to thereby separating it from theculture, and liquefying the separated ethanol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing showing ethanol productivity of the bacterial strainof the present invention.

FIG. 2 is a drawing showing ethanol productivity of the bacterial strainof the present invention by enrichment culture.

FIG. 3 is a drawing showing an outline of the continuous culturingsystem of the present invention.

FIG. 4 is a drawing showing changes of the ethanol production by thecontinuous culturing of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

With the object of solving the aforementioned problems, the inventors ofthe present invention have conducted intensive studies and found as aresult that a specified bacterium produces ethanol at a high temperatureof 40° C. or more from gas such as carbon dioxide as the raw material,and that production of ethanol while continuously recovering the formedethanol from the culture is possible by the use of such bacterium in anethanol production process which uses a gas as the raw material at ahigh temperature, and established the present invention.

The bacterium to be used in the ethanol production process of thepresent invention is a bacterium capable of producing ethanol at atemperature of 40° C. or more (to be referred also to as “ethanolproducing bacterium of the present invention” hereinafter). Completelynothing has not been known so far about a bacterium capable of producingethanol at a high temperature of 40° C. or more. Ethanol productiontemperature of the ethanol producing bacterium of the present inventionis preferably from 50 to 100° C., more preferably from 50 to 80° C.

The ethanol producing bacterium of the present invention is preferablythe one capable of producing ethanol from a carbon compound as the rawmaterial which is gaseous (also called a gas) at ordinary temperatureand ordinary pressure. The term ordinary temperature and ordinarypressure as used herein means 25° C. and atmospheric pressure. Thecarbon compound includes such as carbon monoxide, carbon dioxide,methane, ethane and ethylene, and among these, one or two selected fromcarbon monoxide and carbon dioxide are preferred. In addition to thesecarbon compounds, hydrogen, hydrogen sulfide and the like can also beused together. Examples of the mixed gas include a mixed gas of carbonmonoxide and hydrogen, a mixed gas of carbon dioxide and hydrogen, amixed gas of carbon monoxide, carbon dioxide and hydrogen and the like.In that case other kind of gasses such as nitrogen and the like inertgasses, methane gas and hydrogen sulfide are mixed, they have noparticular problems unless the ethanol production is inhibited. Also,mixing ratio of respective components in the mixed gas is notparticularly limited, unless the ethanol production is inhibited. Inaddition, although it is possible to add these carbon compounds in agaseous state, carbon dioxide may be generated in a culture by addingsubstance which can be used as the carbon dioxide such as a carbonategenerating source. Among these combinations, the use of carbon dioxideand hydrogen has the highest green house gas reducing effect. In thisconnection, the carbon dioxide and carbon monoxide according to thepresent invention include those which are produced by oxidation such asburning of a carbon-containing material, namely gasses such as methaneand ethane, organic material such as coal and wood. In addition, thehydrogen according to the present invention includes those which areproduced by oxidizing hydrogen-containing materials such as hydrogensulfide or methane, as a matter of course.

The ethanol producing bacterium of the present invention is notparticularly limited, with the proviso that it is a bacterium capable ofproducing ethanol at a high temperature condition of 40° C. or more froma carbon compound as the raw material which is gaseous at ordinarytemperature and ordinary pressure, and although it may be either anaerobic bacterium or an anaerobic bacterium, its preferred examplesinclude bacteria belonging to the genus Clostridium or its derivativegenus Thermoanaerobacterium, genus Thermoanaerobacter and genusMoorella, which are classified into CLUSTERS V, VI and VII by the reportof Collins et al. (Collins M. D., International Journal of SystematicBacteriology, October, 812-826 (1994)). In addition, Clostridium sp. No.16-1, Clostridium sp. No. 16-2 and Clostridium sp. No. 22-1, which arebacteria of new species found for the first time by the presentinvention, and analogous bacteria having similar properties to thebacteria as the species can also be-used suitably. Clostridium sp. No.16-1, Clostridium sp. No. 16-2 and Clostridium sp. No. 22-1 have beendeposited on May 2, 2003, as FERM BP-8372, FERM BP-8373 and FERMBP-8374, respectively in International Patent Organism Depositary,National Institute of Advanced Industrial Science and Technology(Central 6, 1-1 Higashi, Tsukuba, Ibaraki, Japan). In this connection,as described in the above, the bacteria found by the present inventionare bacteria of new species belonging to the genus Clostridium, andalthough they also have the properties of the genusThermoanaerobacterium, genus Thermoanaerobacter and genus Moorella whichare derivative species of the genus Clostridium, their variousproperties as the index in determining the genus and species are clearlydifferent from known bacteria of the same genus, so that a possibilityof being new species is remarkably high. In this connection, thebacteria found by the present invention also produce organic acids suchas acetic acid in addition to ethanol.

In addition, mutants of the ethanol producing bacterium of the presentinvention are included within the scope of the present invention asanalogous bacterium, so far as said mutants strain produce ethanol at ahigh temperature condition of 40° C. or more. Examples of the treatmentfor inducing mutation include ultraviolet ray irradiation, X-rayirradiation, radiation exposure, treatment with mutagen such asnitrosoguanidine and acridine dye, or treatment by genetic engineeringtechniques and the like.

According to the culturing process using an anaerobic bacterium,although it is principally the same as the case of generalmicroorganisms, it is necessary to prevent contamination of oxygen, sothat a process to stand still or shake in a culture vessel sealed with abutyl rubber stopper or the like is used in a laboratory. When the scaleis relatively large, a generally used fermentor can be used, and it ispossible to make an anaerobic condition by replacing oxygen in theapparatus with inert gas such as nitrogen or raw material gas such ascarbon dioxide.

In addition to gaseous carbon compounds such as carbon dioxide andcarbon monoxide, inorganic salts which can generate carbon dioxide suchas carbonatescan also be used as the carbon sources to be used in theculturing. However, the carbon sources are not limited to gasses, andraw materials generally used in the culturing such as saccharides andamino acids can also be used together. In this connection, not only theethanol producing bacterium of the present invention can produce ethanolfrom a gas as the raw material, but it also can produce ethanol when asugar is used as the raw material (cf. Example 1).

In addition, there is also no particular limitations too in the gassupplying process, and its examples include a process in whichpressurization is carried out in order to dissolve a gas in the mediumand a process in which a gas is continuously supplied at ordinarypressure. As a process for dissolving in the medium, it is possible toadd salts which can generate gases, such as carbonates and bicarbonates.

As the nitrogen sources, various nitrogen compounds generally useful infermentation, ammonium salts such as ammonium chloride and nitrates suchas sodium nitrate can be used. In addition, components to be added tothe medium are not particularly limited too, and it is a processgenerally used in the culturing to add inorganic compounds such aspotassium dihydrogenphosphate, magnesium sulfate, manganese sulfate,sodium chloride, cobalt chloride, calcium chloride, zinc sulfate, coppersulfate and sodium molybdate and vitamin sources such as yeast extract.

The culturing mode is not particularly limited, and in addition to thegenerally used stirred tank reactor, a single stage or multistage airlift reactor, a dry tube type fermentor, a packing type fermentor, afluidized bed reactor and the like can also be used.

Ethanol is produced by the ethanol production process of the presentinvention by culturing at a high temperature condition of 40° C. or morefrom a gas as the raw material and it is found that its continuousproduction is possible when the ethanol formed by culturing at a hightemperature is vaporized and separated from the culture, since theproduction inhibition by the accumulation of ethanol can be improved andthe vaporized ethanol discharged together with the unreacted gas can beeasily separated and recovered by a cooling or the like means. Inaddition to the aforementioned ethanol production which uses a gas asthe raw material, when a bacterium capable of forming ethanol at a hightemperature condition is used, such a continuous culturing can also beapplied to an ethanol production which uses a conventional sugar or thelike raw material. In this connection, the medium composition in saidcontinuous culturing is the same as described in the foregoing and hasno particular limitation.

In addition, there is no particular limitation about the culturing modein said continuous production. Although it is necessary in general toincrease cell density at the time of culturing in order to carry outefficient production, the conventional processes well known to thoseskilled in the art can be used in the present invention, too. Forexample, in addition to the process shown in the flow chart of FIG. 3, aprocess which uses concentrated cells or a process which usesimmobilized cells can also be used. Examples of the immobilizationprocess include a generally used beads-applied process, amembrane-applied process, a hollow fiber-applied process and the like.In this connection, in the conventional hollow fiber-applied process forthe production of ethanol from a sugar as the raw material, technicalproblem such as clogging due to the raw material has been pointed out,but it is possible to solve this problem by from a gas as the rawmaterial.

Regarding the conditions for vaporizing the formed ethanol, it ispossible under any one of the pressurized, ordinary pressure and reducedpressure conditions, but it is desirable to decide by taking intoconsideration of temperature condition, solubility of the gas and thelike factors from the viewpoint of continuous nature of the culturing.

Regarding the method for liquefying vaporized ethanol, there is noparticular limitation, and conventionally known cooling methods can beemployed, such as a cooling which uses water as the coolant, a coolingwhich uses air as the coolant and a method which uses a gas as thecoolant. In addition, there is no particular limitation regarding thecooling temperature, so long as it is a temperature at which thevaporized ethanol is liquefied, and it is desirable to carry out it at atemperature of the boiling point or less of ethanol (78° C. or less) inthe case of ordinary pressure.

The present invention is described in the following based onillustrative examples, but the present invention is not restricted bythese examples.

EXAMPLE 1

Although there are thermophilic acetic acid bacteria in theconventionally known congeneric species or sibling species, it isconsidered that the bacteria found by the present invention are newbacterial species since they are different from the conventionally knownbacteria of the same genus in terms of their ability to produce ethanoland other various properties which are described in the following. Amongthe found bacterial strains, taxonomic properties of strains No. 16-1,No. 16-2 and No. 22-1 are described. Since formal names are not givenyet, they are expressed in the present invention as Clostridium sp. No.16-1 (FERM BP-8372), Clostridium sp. No. 16-2 (FERM BP-8373) andClostridium sp. No. 22-1 (FERM BP-8374). Examination of thebacteriological properties was carried out in accordance with the methoddescribed in “Classification and Identification of Microorganisms (Thelast volume)” (edited by T. Hasegawa, published by Gakkai Shuppan Center(written in Japanese)) and Bergey's Manual of Systematic Bacteriology(1984). In addition, estimation of the belonging taxon was carried outby examining about 500 bp of partial nucleotide sequence of 16S rDNA.

(1) Taxonomical Properties of No. 16-1

Creation Method:

This strain is a bacterial strain isolated from a soil sample collectedin Shiobara, Tochigi, Japan. 5 ml of the liquid medium shown in Table 1was dispensed into test tubes, and after sterilization, about 0.5 g ofeach soil sample was added. The tube was sealed with a butyl rubberstopper, and then the gas phase was replaced by a gas containinghydrogen (75%) and carbon dioxide (25%), followed by subculture at aninterval of 3 weeks at 55° C. on a shaker. After carrying out thesubculture twice with the liquid medium, single colony was isolated by aroller tube method (Methods in Microbiology, Vol. 3 B, paragraph 117(1969), Academic Press) using an agar medium prepared by adding 0.5%fructose and 2% agar, followed by culturing at 55° C. on a shaker usingthe liquid medium shown in Table 1 in which the gas phase was replacedby a gas containing hydrogen (75%) and carbon dioxide (25%) to obtainthe strain.

Microscopic Observation:

-   1. Shape and size of cell: single rod or double rods (curved), 0.5    μm in width, 3.0 to 4.0 μm in length-   2. Flagella: exist-   3. Spore: exist-   4. Gram staining: positive (irregular in later stage of culturing)    Medium Composition:

Exemplified in Table 1. TABLE 1 Composition of basal medium (in 1 literof distilled water) NH₄Cl 1.0 g KCl 0.1 g MgSO₄.7H₂O 0.2 g NaCl 0.8 gKH₂PO₄ 0.1 g CaCl₂.2H₂O 0.02 g Yeast extract 1.0 g NaHCO₃ 2.0 g Mineralsolution (1) 10 ml Vitamin solution (2) 10 ml Resazurin solution (0.1%)1 ml Reducing solution (3) 10 ml pH 6.5 (1) Mineral solution (in 1 literof distilled water) Nitrilotriacetic acid 2.0 g (adjusted to pH 6.0 withKOH) MnSO₄.H₂O 1.0 g Fe(SO₄)₂(NH₄)₂.6H₂O 0.8 g CoCl₂.6H₂O 0.2 gZnSO₄.7H₂O 0.2 g CuCl₂.2H₂O 0.02 g NiCl₂.6H₂O 0.02 g Na₂MoO₄.2H₂O 0.02 gNa₂SeO₄ 0.02 g Na₂WO₄ 0.02 g (2) Vitamin solution (in 1 liter ofdistilled water) Biotin 2.0 mg Folic acid 2.0 mg Pyridoxinehydrochloride 10.0 mg Thiamine hydrochloride 5.0 mg Riboflavin 5.0 mgNicotinic acid 5.0 mg Calcium pantothenate 5.0 mg Vitamin B₁₂ 0.1 mgp-Aminobenzoic acid 5.0 mg Thioctic acid 5.0 mg (3) Reducing solution(in 0.1 liter of distilled water) Cysteine hydrochloride monohydrate 4.0g Sodium sulfide nonahydrate 4.0 g Sodium hydroxide 0.9 gGrowth Conditions:

Growth on an agar medium prepared by adding 2% of agar to thecomposition of Table 1 is as follows.

Shape: circular

Fringe: smooth

Upheaval: slightly raised

Surface: smooth, lustrous

Color tone: cream color

Physiological Properties:

Behavior against oxygen: obligate anaerobic

Growth pH range: optimum pH 7.0, growth pH range 5.0 to 8.0

Growth temperature range: optimum temperature 60° C., growth range 40 to65° C.

Indole production: −

Hydrolysis of gelatin: +

Hydrolysis of aesculin: +

Catalase production: −

Formation of pigment: −

Vitamin requirement: thiamine

Assimilation of Carbon Sources and the Like:

Tests on biochemical properties were carried out by an API system(bioMerieux France) in accordance with the measuring method of API 20A.In addition, assimilation of carbon sources was verified by carrying outthe following test as an additional test.

With 1% of each carbon source, 5 ml of the medium of Table 1 wassupplemented and put into a test tube, and the strain was inoculatedinto the thus prepared aseptic medium, followed by stationary culturesat 60° C. for 7 days after replacing the gas phase with sterilizednitrogen gas. Regarding the growth, absorbance at 660 nm was measuredusing a spectrophotometer (U 2100 PC, manufactured by Shimadzu). Whendifference in the absorbance at 660 nm from that of the control whichcontains no carbon source was less than 0.1, it was judged as “notassimilated”, 0.1 or more and less than 0.3 as “slightly assimilated”,and 0.3 or more as “assimilated”.

“Assimilated”

D-glucose, D-fructose, galactose, D-xylose, arabinose, trehalose,mannitol, lactose, maltose, salicin, D-cellobiose and D-mannose.

In addition, it also assimilated carbon dioxide under an environment ofthe presence of carbon monoxide and hydrogen.

“Slightly assimilated”

Rhamnose and ribose.

“Not assimilated”

Sorbitol, glycerin and D-raffinose.

Products:

With 1% of each carbon source whose assimilation was confirmed, 5 ml ofthe medium of Table 1 was supplemented and put into a test tube, and thestrain was inoculated into a prepared aseptic medium followed bystationary culture at 60° C. for 7 days after replacing the gas phasewith sterilized nitrogen gas. Production of ethanol and acetic acid wasconfirmed in all of-the carbon sources.

Also, when carbon dioxide was used as the carbon source, 5 ml of themedium of Table 1 was put into a test tube, and the strain wasinoculated into a prepared aseptic medium followed by shaking culture at55° C. for 7 days after replacing the gas phase with a sterilized gas ofcarbon dioxide (25%) and hydrogen (75%). In that case, production ofethanol and acetic acid was confirmed.

Partial Nucleotide Sequence of 16S rDNA:

Genomic DNA was extracted from the strain using PrepMan (registeredtrademark) Method (Applied Biosystems, US). Using the thus extractedgenomic DNA as the template, a partial nucleotide sequence of about 500bp of the 16S rDNA was amplified by PCR, and the nucleotide sequence wassubjected to sequencing and used for the analysis. MicroSeq (registeredtrademark) 500 16S rDNA Bacterial Sequencing Kit (Applied Biosystems,US) was used in the purification and cycle sequencing of the PCRproduct. With regard to the operations from the genomic DNA extractionto cycle sequencing, they were carried out in accordance with theprotocol (P/N 4308132 Rev. A) of Applied Biosystems, and GeneAmp PCRSystem 9600 (Applied Biosystems, US) was used as the thermal cycler,while ABI PRISM 377 DNA Sequencer (Applied Biosystems, US) was used asthe DNA Sequencer.

The partial nucleotide sequence of 16S rDNA of the strain was thenucleotide sequence shown in SEQ ID NO;1.

When homology search of the thus obtained 16S rDNA was carried out for aDNA nucleotide sequence data base (GenBank/EMBL/DDBJ) using BLAST,homology was 98.34% with Thermoanaerobacterium aotearoense JW/SL-NZ613T,97.18% with Thermoanaerobacterium sp. C-1, 97.18% withThermoanaerobacterium sp. C38-4, 96.97% with Clostridium sp. C-4 and95.79% with Clostridium sp. C41-3, so that it was considered that itbelongs to the genus Clostridium.

Comparison with Conventional Analogous Species and the Like:

Based on the aforementioned bacteriological properties, No. 16-1 is astrain of obligate anaerobic spore forming rod which is characterized bythe production of ethanol and acetic acid as the main fermentationproducts from carbon dioxide and hydrogen. When these properties aresearched with reference to Bergey's Manual of DeterminativeBacteriology, 8th edition, Bergey's Manual of Systematic Bacteriologyand Classification and Identification of Microorganisms (the lastvolume), the strain is considered to be a strain belonging to the genusClostridium. In addition, there are no descriptions in Bergey's Manualof Determinative Bacteriology, 8th edition, Bergey's Manual ofSystematic Bacteriology, and Handbook of Extreme EnvironmentalMicroorganisms (T. Oshima, Science Forum 1991), of bacterial specieswhose various properties coincide with those of No. 16-1. As bacteriawhich grow at a high temperature condition of 50° C. or more and produceacetic acid by growing on carbon dioxide and hydrogen, Moorellathermoacetica (Clostridium thermoaceticum), Moorella thermoautotrophica(Clostridium thermoautotrophicum) and Acetogenium kivui are known, butbacteria which produce ethanol and acetic acid from the same materialshave not been found yet. In addition, when its properties were comparedwith those of the bacteria, they were different in terms of the pointsshown in Table 2.

Since this strain was considered to be a new species belonging to thegenus Clostridium based on the above results, this was named Clostridiumsp. No. 16-1.

(2) Taxonomical Properties of No. 16-2 Creation Method:

This strain is a bacterial strain isolated from a soil sample collectedin Shiobara, Tochigi, Japan. Into test tubes, 5 ml of the liquid mediumshown in Table 1 was dispensed, and after sterilization, 0.5 g of eachsoil sample was added. The tube was sealed with a butyl rubber stopper,and then the gas phase was replaced by a gas containing hydrogen (75%)and carbon dioxide (25%), followed by subculture at an interval of 3weeks at 55° C. on a shaker. After carrying out the subculture twicewith the liquid medium, single colony was isolated by the roller tubemethod using an agar medium prepared by adding 0.5% fructose and 2%agar, followed by culturing at 55° C. on a shaker using the liquidmedium shown in Table 1 in which the gas phase was replaced by a gascontaining hydrogen (75%) and carbon dioxide (25%) to obtain the strain.

Microscopic Observation:

-   1. Shape and size of cell: single rod or double rods (curved), 0.6    to 0.7 μm in width, 3.0 to 5.0 μm in length-   2. Flagella: exist-   3. Spore: exist-   4. Gram staining: positive (irregular in later stage of culturing)    Medium Composition:

Exemplified in Table 1.

Growth Conditions:

Growth on an agar medium prepared by adding 2% of agar to thecomposition of Table 1 is as follows.

Shape: circular

Fringe: smooth

Upheaval: slightly raised

Surface: smooth, lustrous

Color tone: cream color

Physiological Properties:

Behavior against oxygen: obligate anaerobic

Growth pH range: optimum pH 7.0, growth pH range 5.0 to 8.0

Growth temperature range: optimum temperature 60° C., growth range 45 to65° C.

Indole production: −

Hydrolysis of gelatin: +

Hydrolysis of aesculin: +

Catalase production: −

Formation of pigment: −

Vitamin requirement: no

Assimilation of Carbon Sources and the Like:

Tests on biochemical properties were carried out by an API system(biomerieux France) in accordance with the measuring method of API 20A.In addition, assimilation of carbon sources was verified by carrying outthe following test as an additional test.

Into a test tube, 5 ml of the medium of Table 1 supplemented with 1% ofeach carbon source was added, and the strain was inoculated into thethus prepared aseptic medium, followed by stationary culture at 60° C.for 7 days after replacing the gas phase with sterilized nitrogen gas.With regard to the growth, absorbance at 660 nm was measured using aspectrophotometer (UV 2100 PC, manufactured by Shimadzu). Whendifference in the absorbance at 660 nm from that of the control whichcontains no carbon source was less than 0.1, it was judged as “notassimilated”, 0.1 or more and less than 0.3 as “slightly assimilated”,and 0.3 or more as “assimilated”.

“Assimilated”

D-glucose, D-fructose, galactose, xylose, arabinose, trehalose,D-mannitol, lactose, maltose, salicin, glycerin, cellobiose, D-mannoseand D-raffinose.

In addition, it also assimilated carbon dioxide under an environment ofthe presence of carbon monoxide and hydrogen.

“Slightly Assimilated”

Rhamnose.

“Not Assimilated”

Ribose and sorbitol.

Products:

Into a test tube, 5 ml of the medium of Table 1 supplemented with 1% ofeach carbon source whose assimilation was confirmed was added, and thestrain was inoculated into the prepared aseptic medium, followed bystationary culture at 60° C. for 7 days after replacing the gas phasewith sterilized nitrogen gas. Production of ethanol and acetic acid wasconfirmed in each of the carbon sources.

Also, when carbon dioxide was used as the carbon source, 5 ml of themedium of Table 1 was put into a test tube, and the strain wasinoculated into the prepared aseptic medium, followed by shaking cultureat 55° C. for 7 days after replacing the gas phase with a sterilized gasof carbon dioxide (25%) and hydrogen (75%). In that case, production ofethanol and acetic acid was confirmed.

Partial Nucleotide Sequence of 16S rDNA:

Genomic DNA was extracted from the strain using PrepMan (registeredtrademark) Method (Applied Biosystems, US). Using the thus extractedgenomic DNA as the template, a nucleotide sequence of about 500 bp ofthe 16S rDNA was amplified by PCR, and the nucleotide sequence wassubjected to sequencing and used in the analysis. MicroSeq (registeredtrademark) 500 16S rDNA Bacterial Sequencing Kit (Applied Biosystems,US) was used for the purification and cycle sequencing of the PCRproduct. With regard to the operations from the genomic DNA extractionto cycle sequencing, they were carried out in accordance with theprotocol (P/N 4308132 Rev. A) of Applied Biosystems, and GeneAmp PCRSystem 9600 (Applied Biosystems, US) was used as the thermal cycler, andABI PRISM 377 DNA Sequencer (Applied Biosystems, US) as the DNASequencer.

The partial nucleotide sequence of 16S rDNA of this strain was thenucleotide sequence shown in SEQ ID NO;2.

When homology search of the thus obtained 16S rDNA was carried out for aDNA nucleotide sequence data base (GenBank/EMBL/DDBJ) using BLAST, itwas 99.08% with Moorella thermoautotrophica (Clostridiumthermoautotrophicum) DSM1974, 98.90% with Moorella thermoacetica(Clostridium thermoaceticum) ATCC 39037, and 98.71% with Moorellathermoacetica (Clostridium thermoaceticum) ET-5a, so that it wasconsidered that the strain belongs to the genus Clostridium.

Comparison with Conventional Analogous Species and the Like:

Based on the aforementioned bacteriological properties, No. 16-2 is astrain of obligate anaerobic rod which is characterized by theproduction of ethanol and acetic acid as the main fermentation metabolicproducts from carbon dioxide and hydrogen. When these properties areretrieved with reference to Bergey's Manual of DeterminativeBacteriology, 8th edition, Bergey's Manual of Systematic Bacteriologyand Classification and Identification of Microorganisms (the lastvolume), this is considered to be a strain belonging to the genusClostridium. In addition, there are no descriptions in Bergey's Manualof Determinative Bacteriology, 8th edition, Bergey'Manual of SystematicBacteriology, and Handbook of Extreme Environmental Microorganisms (T.Oshima, Science Forum 1991), on bacterial species whose variousproperties coincide with those of No. 16-1. As bacteria which grow at ahigh temperature condition of 50° C. or more and produce acetic acid bygrowing on carbon dioxide and hydrogen, Moorella thermoacetica(Clostridium thermoaceticum), Moorella thermoautotrophica (Clostridiumthermoautotrophicum) and Acetogenium kivui are known. However, there areno reports which states that the bacteria produce ethanol and aceticacid. In addition, when its properties were compared with those of thebacteria, they were different in terms of the points shown in Table 2.

Since this strain was considered to be a new species belonging to thegenus Clostridium based on the above results, the strain was namedClostridium sp. No. 16-2.

(3) Taxonomical Properties of No. 22-1

Creation Method:

This strain is a bacterial strain isolated from a subterranean soilsample collected in Chiba, Japan. That is, 5 ml of the liquid mediumshown in Table 1 was dispensed into test tubes, and after sterilization,0.5 g of each soil sample was added. The tube was sealed with a butylrubber stopper, and then the gas phase was replaced by a gas containinghydrogen (75%) and carbon dioxide (25%) to carry out subculture at aninterval of 3 weeks by culturing at 55° C. on a shaker. After carryingout the subculture twice with the liquid medium, single colony wasisolated by the roller tube method using an agar medium prepared byadding 0.5% fructose and 2% agar, followed by culturing the strain at55° C. on a shaker using the liquid medium shown in Table 1 in which thegas phase was replaced by a gas containing hydrogen (75%) and carbondioxide (25%) to obtain the strain.

Microscopic Observation:

-   1. Shape and size of cell: single rod or double rods, 0.5 to 0.6 μm    in width, 2.0 to 3.0 μm in length-   2. Flagella: exist-   3. Spore: exist-   4. Gram staining: positive    Medium Composition:

Exemplified in Table 1.

Growth Conditions:

Growth on an agar medium prepared by adding 2% of agar to thecomposition of Table 1 is as follows.

Shape: circular

Fringe: smooth

Upheaval: slightly raised

Surface: smooth, lustrous

Color tone: cream color

Physiological Properties:

Behavior against oxygen: obligate anaerobic

Growth pH range: optimum pH 7.0, growth pH range 5.0 to 7.5

Growth temperature range: optimum temperature 60° C., growth range 40 to75° C.

Indole production: −

Hydrolysis of gelatin: +

Hydrolysis of aesculin: +

Catalase production: −

Formation of pigment: −

Vitamin requirement: no

Assimilation of Carbon Sources and the Like:

Tests on biochemical properties were carried out-by an API system(bioMerieux France) in accordance with the measuring method of API 20A.In addition, assimilation of carbon sources was verified by carrying outthe following test as an additional test.

In a test tube, 5 ml of the medium of Table 1 supplemented with 1% ofeach carbon source was added, and the strain was inoculated into theaseptic medium, followed by stationary culture at 60° C. for 7 daysafter replacing the gas phase with sterilized nitrogen gas. With regardto the growth, absorbance at 660 nm was measured using aspectrophotometer (UV 2100 PC, manufactured by Shimadzu). Whendifference in the absorbance at 660 nm from that of the controlcontaining no carbon source was less than 0.1, it was judged as “notassimilated”, 0.1 or more and less than 0.3 as “slightly assimilated”,and 0.3 or more as “assimilated”.

“Assimilated”

D-glucose, D-fructose, galactose, xylose, arabinose, trehalose, ribose,lactose, maltose, salicin, D-cellobiose and D-mannose.

In addition, it also assimilated carbon dioxide under an environment ofthe presence of carbon monoxide and hydrogen.

“Slightly assimilated”

Rhamnose.

“Not assimilated”

Sorbitol, glycerin and raffinose.

Products:

In a test tube, 5 ml of the medium of Table 1 supplemented with 1% ofeach carbon source whose assimilation was confirmed was added, and thestrain was inoculated into the prepared aseptic medium, followed bystationary culture at 60° C. for 7 days after replacing the gas phasewith sterilized nitrogen gas. Production of ethanol and acetic acid wasconfirmed in each of the carbon sources.

Also, when carbon dioxide was used as the carbon source, 5 ml of themedium of Table 1 was added in a test tube, and the strain wasinoculated into the prepared aseptic medium, followed by shaking cultureat 55° C. for 7 days after replacing the gas phase with a sterilized gasof carbon dioxide (25%) and hydrogen (75%). In that case, production ofethanol and acetic acid was confirmed.

Partial Nucleotide Sequence of 16S rDNA:

Genomic DNA was extracted from the strain using PrepMan (registeredtrademark) Method (Applied Biosystems, US). Using the thus extractedgenomic DNA as the template, a nucleotide sequence of about 500 bp ofthe 16S rDNA was amplified by PCR, and the nucleotide sequence wassubjected to sequencing and used for the analysis. MicroSeq (registeredtrademark) 500 16S rDNA Bacterial Sequencing Kit (Applied Biosystems,US) was used in the purification and cycle sequencing of the PCRproduct. With regard to the operations from the genomic DNA extractionto cycle sequencing, they were carried out in accordance with theprotocol (P/N 4308132 Rev. A) of Applied Biosystems, and GeneAmp PCRSystem 9600 (Applied Biosystems, US) was used as the thermal cycler, andABI PRISM 377 DNA Sequencer (Applied Biosystems, US) as the DNASequencer.

The partial nucleotide sequence of 16S rDNA of the strain was thenucleotide sequence shown in SEQ ID NO;3.

When homology search of the thus obtained 16S rDNA was carried out for aDNA nucleotide sequence data base (GenBank/EMBL/DDBJ) using BLAST, itshowed a homology of 98.14% with Thermoanaerobacterium aotearoenseJW/SL-NZ613T, 97.58% with Clostridium thermoamylolyticum DMS2335 and97.18% with Thermoanaerobacterium sp. c38-4, so that it was consideredthat the strain belongs to the genus Clostridium.

Comparison with Conventional Analogous Species and the Like:

Based on the aforementioned bacteriological properties, No. 16-2 is astrain of obligate anaerobic rod which is characterized by theproduction of ethanol and acetic acid as the main fermentation productsfrom carbon dioxide and hydrogen. When the properties are retrieved withreference to Bergey's Manual of Determinative Bacteriology, 8th edition,Bergey's Manual of Systematic Bacteriology and Classification andIdentification of Microorganisms (the last volume), this is consideredto be a strain belonging to the genus Clostridium. In addition, there isno description in Bergey's Manual of Determinative Bacteriology, 8thedition, Bergey's Manual of Systematic Bacteriology, and Handbook ofExtreme Environmental Microorganisms (T. Oshima, Science Forum 1991), onbacterial species whose various properties coincide with those of No.22-1. As bacteria which grow at a high temperature condition of 50° C.or more and produce acetic acid by growing on carbon dioxide andhydrogen, Moorella thermoacetica (Clostridium thermoaceticum), Moorellathermoautotrophica (Clostridium thermoautotrophicum) and Acetogeniumkivui are known, but there are no reports stating that the bacteriaproduce ethanol and acetic acid (Table 2). Also, although the partialnucleotide sequence of 16S rDNA of the strain was similar to that of No.16-1, they were distinguishable based on their differences in the cellshape and vitamin requirement. In addition, when its properties werecompared with those of the bacteria, they were different in terms of thepoints shown in Table 2.

Since the strain was considered to be a new species belonging to thegenus Clostridium based on the above results, it was named Clostridiumsp. No. 22-1. TABLE 2 Moorella Moorella thermoacetica thermoautotrophicaClostridium sp. Clostridium sp. Clostridium sp. (C. thermoaceticum) (C.thermoautotrophicum) No. 16-1 No. 16-2 No. 22-1 (reported data)(reported data) (actual value) (actual value) (actual value) Microscopicobservation Shape of cell Rod Rod Rod Rod Rod (curved) (curved)Physiological property Growth pH 5.0-7.0 4.7-7.5 5.0-8.0 5.0-8.0 5.0-7.5Growth temperature 45-65 36-70 40-65 45-65 40-80 Hydrolysis of gelatin −− + + + Hydrolysis of aesculin − − + + + Assimilation of C source ribose− + ± − + arabinose − − + + + rhamnose − + ± − − trehalose − − + + +Production from CO₂/H₂ Ethanol − − + + + Acetic acid + + + + +

EXAMPLE 2

Each of Clostridium sp. No. 16-1, Clostridium sp. No. 16-2 andClostridium sp. No. 22-1 obtained in the present invention was culturedin the following manner. Into test tubes, 5 ml of the medium shown inTable 1 was dispensed and sterilized, and the gas phase was replaced bya sterilized gas containing carbon dioxide (25%) and hydrogen (75%),followed by adjusting to 2 atmospheric pressure with the same gas. 250μl of the culture broth obtained by culturing each strain using the samemedium was added thereto using a sterilized syringe and cultured at 55°C. for 10 days with shaking at 150 rpm. A portion of the culture brothwas centrifuged to separate cells and the product was determined by gaschromatography. As a result, 0.5 mM of ethanol was produced byClostridium sp. No. 16-1, 1.8 mM of the same by Clostridium sp. No. 16-2and 1.5 mM of the same by Clostridium sp. No. 22-1.

EXAMPLE 3

Clostridium sp. No. 16-2 was cultured in the following manner. Into testtubes, 5 ml of the medium shown in Table 1 was dispensed. Aftersterilization, the gas phase was replaced by a sterilized gas containingcarbon monoxide (60%), carbon dioxide (10%) and hydrogen (30%) (to bereferred to as gas A hereinafter), or a sterilized gas containing carbondioxide (25%) and hydrogen (75%) (to be referred to as gas Bhereinafter) followed by adjusting to 2 atmospheric pressure with eachgas. 250 μl of the culture broth obtained by culturing the strain usingthe same medium was added thereto using a sterilized syringe andcultured at 55° C. with shaking at 150 rpm. A portion of the culturebroth on the 7th or 14th day was treated with a centrifuge to separatecells and to carry out determination of the product by gaschromatography. As a result, the maximum ethanol production was 3.8 mMwhen Clostridium sp. No. 16-2 was cultured using the gas A (FIG. 1).

EXAMPLE 4

An enrichment culturing was carried out using Clostridium sp. No. 16-2.Stationary culture was carried out at 60° C. for 3 days using a mediumprepared by adding 0.5% fructose to the medium shown in Table 1, and theresulting cells were recovered by centrifugation. Into test tubes, 5 mlof the medium shown in Table 1 was dispensed. After sterilization, thegas phase was replaced by a sterilized gas containing carbon dioxide(25%) and hydrogen (75%), followed by adjusting to 2 atmosphericpressure with the same gas. The cells adjusted to an absorbance at 660nm to be 4.0 were added thereto using a sterilized syringe and culturedat 55° C. with shaking at 150 rpm. A portion of the culture broth on the7th or 14th day was centrifuged to separate cells and to carry outdetermination of the product by gas chromatography. As a result, ethanolproduction was confirmed at the maximum of 11 mM (FIG. 2).

EXAMPLE 5

Continuous culture was carried out using concentrated cells ofClostridium sp. No. 16-2. Stationary culture was carried out at 60° C.for 5 days using a medium prepared by adding 0.5% fructose to the mediumshown in Table 1, and the resulting cells were recovered bycentrifugation. Into a 1 liter capacity culture vessel, a 300 ml portionof the medium shown in Table 1 was added and sterilized, and then thegas phase was replaced with a sterilized gas containing carbon dioxide(25%) and hydrogen (75%). The recovered cells were prepared such thatthe absorbance at 660 nm became 4.0, and added to the culture vesselusing a syringe, followed by shaking culture at 60° C. and ordinarypressure at 800 rpm using the apparatus shown in FIG. 3. The solutionobtained from the outlet of condenser using water of 20° C. wasrecovered, and determination of ethanol was carried out by gaschromatography. As a result of 28 days of the continuous culturing, itwas able to maintain an average production of 1.0 g/liter/day (FIG. 4).

The present invention is precisely explained with reference to specificembodiments but it is apparent for those skilled in the art that variouschanges and modifications can be carried out without departing from thespirit and scope of the present invention.

The present application is based on Japanese Patent ApplicationNo.2002-155085 filed on May 29, 2002 and the entire content isincorporated herein by reference.

INDUSTRIAL APPLICABILITY

According to the present invention, a bacterium which can efficientlyproduces ethanol from gasses such as carbon dioxide as the raw materialat a high temperature of 40° C. or more can be provided. In addition, bythe use of the bacterium, it is possible that inexpensive, efficient andcontinuous production of ethanol from gasses such as carbon dioxide as acause of the global warming, directly without cooling.

1. A bacterium capable of producing ethanol at a temperature of 40° C.or more from a carbon compound which is gaseous at ordinary temperatureand ordinary pressure as the raw material.
 2. The bacterium according toclaim 1, wherein the carbon compound is at least one selected from thegroup consisting of carbon monoxide, carbon dioxide, methane, ethane andethylene.
 3. The bacterium according to claim 1 or 2, wherein the carboncompound is at least one selected from the group consisting of carbonmonoxide and carbon dioxide.
 4. The bacterium according to any one ofclaims 1 to 3, which belongs to the genus Clostridium or a derivativegenus thereof.
 5. The bacterium according to claim 4, wherein the genusClostridium or a derivative genus thereof is the genus Clostridium, thegenus Thermoanaerobacterium, the genus Thermoanaerobacter or the genusMoorella.
 6. The bacterium according to any one of claims 1 to 5, whichbelongs to the genus Clostridium.
 7. The bacterium according to any oneof claims 1 to 6, which is Clostridium sp. No. 16-1 (FERM BP-8372),Clostridium sp. No. 16-2 (FERM BP-8373), Clostridium sp. No. 22-1 (FERMBP-8374) or an analogous bacterium thereof.
 8. A process for producingethanol, which comprises culturing, at a temperature of 40° C. or more,a bacterium capable of producing ethanol at a temperature of 40° C. ormore from a carbon compound which is gaseous at ordinary temperature andordinary pressure as the raw material.
 9. The process according to claim8, wherein said culturing is carried out in the presence of a carboncompound which is gaseous at ordinary temperature and ordinary pressure.10. The process according to claim 8 or 9, wherein the carbon compoundis at least one selected from the group consisting of carbon monoxide,carbon dioxide, methane, ethane and ethylene.
 11. The process accordingto any one of claims 8 to 10, wherein the carbon compound is at leastone selected from the group consisting of carbon monoxide and carbondioxide.
 12. The process according to any one of claims 8 to 11, whereinthe bacterium belongs to the genus Clostridium or a derivative genusthereof.
 13. The process according to claim 12, wherein the genusClostridium or a derivative genus thereof is the genus Clostridium, thegenus Thermoanaerobacterium, the genus Thermoanaerobacter or the genusMoorella.
 14. The process according to any one of claims 8 to 13,wherein the bacterium belongs to the genus Clostridium.
 15. The processaccording to any one of claims 8 to 14, wherein the bacterium isClostridium sp. No. 16-1 (FERM BP-8372), Clostridium sp. No. 16-2 (FERMBP-8373), Clostridium sp. No. 22-1 (FERM BP-8374) or an analogousbacterium thereof.
 16. The process according to any one of claims 8 to15, which further comprises: vaporizing the produced ethanol to therebyseparate it from the culture; and liquefying the separated ethanol. 17.A process for producing ethanol, which comprises: culturing, at atemperature of 40° C. or more, a bacterium capable of producing ethanolat a temperature of 40° C. or more from a carbon compound which isgaseous at ordinary temperature and ordinary pressure as the rawmaterial; vaporizing the produced ethanol to thereby separating it fromthe culture, and liquefying the separated ethanol.